For these experiments we used adult wild type (WT) Oregon-R (Oregon-R-C) and PTEN-induced putative kinase 1 PINK1^B9 (w[*] Pink1[B9]) mutant Drosophila melanogaster (Dm) males (from Bloomington Stock Center; Fly Base: http://flybase.bio.indiana.edu). After emergence from pupae, male WT and PINK1^B9 mutant flies were separated. WT and mutant flies were reared on a standard cornmeal-yeast-agar medium in controlled environmental conditions (24–25°C; 60% relative humidity; light/dark = 12/12 hours). In detail, four groups of mutant flies were reared on a standard medium supplemented with Mucuna pruriens methanolic extract (Mpe) (Batch no. FMPEX/2012060001; Natural Remedies Ltd., Bangalore, India). PINK1^B9 mutants were supplied with Mpe at different concentrations (0 (i.e. untreated PINK1^B9 mutants), 0.1, 1 and 10% w/w in their standard diet) both as larvae and adults (L⁺/A⁺). In addition, another group was reared on a standard medium supplemented with 0.01% (0.5 mM) L-Dopa (Sigma Aldrich, Milan, Italy), a percentage similar to that at which L-Dopa was supplemented with 0.1% Mpe [15]. The effects of Mpe were assayed at different age steps (I: 3–6; II: 10–15; III: 20–25 days old). A series of experiments on life span, using various concentrations of Mpe (see below in Survival curves) provided the basis for selecting the optimal concentration at which conduct the behavioral, morphological, and protein expression assessments. In particular, based on lifespan results, the olfaction behavior assessments, transmission electron microscopy (TEM) and western blot analyses were restricted to group II flies after 0.1% Mpe administration as L⁺/A⁺. Standard genetic procedures were used during the study.

With the aim of selecting the optimal Mpe’s concentration, Dm were grown on standard diet supplemented with different concentrations of Mpe at 25°C. Cohorts of 40 flies (4 flies/tube) from each experimental group (i.e. WT, untreated and Mpe-treated PINK1^B9) were monitored every 2 days for their survival. Mortality was analyzed using Kaplan-Meier survival curves and the statistical comparisons were made with a Gehan-Breslow-Wilcoxon test. Experiments were done in duplicate with the exception of those on WT, untreated mutants, 0.1% Mpe- and 0.01% L-Dopa-treated PINK1^B9 that were done in triplicate.

Each experiment was conducted with the appropriate control group (i.e. WT, untreated PINK1^B9 and treated PINK1^B9).

The climbing assay (negative geotaxis assay) was used to assess locomotor ability [17]. Climbing data were obtained from groups I–III of untreated WT, untreated PINK1^B9 and, as L⁺/A⁺, 0.1, 1 and 10% Mpe- and 0.01% L-Dopa-treated PINK1^B9 mutants. Cohorts of 30 flies from each experimental group were subjected to the assay. Flies were placed individually in a vertically-positioned plastic tube (length 10 cm; diameter 1.5 cm) and tapped to the bottom. Climbing time was recorded upon crossing a line drawn at 6 cm from the bottom. The number of flies that could climb unto, or above, this line within 10 seconds was recorded and expressed as percentage of total flies. Data were expressed as average + SEM from at least three separate experiments. The statistical evaluation was made by two-way ANOVA (p<0.05) followed by HSD post-hoc test.

In vivo electroantennogram recordings (EAG) were performed following a previously described protocol [4]. Briefly, live adult WT Dm and untreated, Mpe- and L-Dopa-treated PINK1^B9 from group II (n = 12/each strain) were singly positioned under the view of an Olympus BX51WI light microscope (Olympus, Tokyo, Japan). Electrodes were silver wires inserted in glass capillaries filled with a saline solution (NaCl 150 mM).The recording glass electrode was positioned on the tip of the left antenna while the reference was pierced through the compound eye. The EAG signal was amplified with an AC/DC probe and then acquired with an IDAC-4 interface board (Syntech, Hilversum, NL). The antennae were constantly blown by a flow of charcoal purified and humidified air (speed 0.5 m/s) via a glass tube. Odor stimuli were administered by injecting a puff of purified air (0.5 s at 10 mL/s airflow) through the pipette using the stimulus delivery controller (Syntech, Hilversum, NL).

Odor stimuli were prepared in 3 step-dose concentration (0.01, 0.1, and 1% v/v) diluted in hexane. Odor stimulus, 1-hexanol, was chosen according to Fishilevich and Vosshall [20], for its well-known stimulant activity in Dm. Mean values of EAG amplitude were calculated and then analyzed by comparing the results obtained in untreated PINK1^B9, Mpe- and L-Dopa-treated flies with matched WT. The significance of differences was tested by one-way ANOVA (followed by HSD post hoc test) with a threshold level of statistical significance set at p<0.05. EAG results are expressed as average values ± S.E.M and represented by histograms.

Free-walking bioassay was performed following the experimental procedures used by Dekker et al. [18]. In particular, group II WT, untreated PINK1^B9 and, as L⁺/A⁺, 0.1% Mpe- and 0.01% L-Dopa-treated PINK1^B9 mutants were given the opportunity to choose between vials containing water with or without odor. Two 4 mL glass vials were placed symmetrically in a large petri dish (arena) and then fitted with truncated pipette tips. The vials were filled with 300 µL of water with 0.25% Triton X (Sigma-Aldrich, Milan, Italy) with or without the odorant (0.1% (v/v) 1-hexanol; Sigma-Aldrich, Milan, Italy). As mentioned above, in order to allow detection of possible Mpe’s effects independently from the circuit, octopaminergic -for appetitive- and dopaminergic -for aversive stimuli [19], 1-hexanol was chosen, according to Fishilevich and Vosshall [20], because the mechanism(s) of olfactory transduction signal involve several glomeruli and complex neural pathways. Flies were starved for 8 hours prior to starting the experiments. These, done in triplicate, were performed in controlled environmental conditions (n = 12 bioassays/each experimental group of flies; n = 20 flies/arena). The assays lasted 18 hours, a streamlined range of time to overtake the possible influence of motor impairment in mutants. The dehydration of flies was prevented by placing a cotton ball with 3 mL of water in the arenas. Data obtained were expressed as average of percentages of flies reaching the 1-hexanol or water trap and statistically evaluated by one-way ANOVA (p<0.05) followed by HSD post-hoc test.

Group II WT, untreated PINK1^B9 and, as L⁺/A⁺, 0.1% Mpe-treated PINK1^B9 mutants were anesthetized using carbon dioxide and carefully decapitated. The brains and the thoracic ganglia, once rapidly removed, were fixed in a mixture of 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, washed several times in the same buffer, post-fixed in 1% osmium tetroxide in distilled water for 2 hours, and stained overnight at 4°C in an aqueous 0.5% uranyl acetate solution. After several washes in distilled water, the samples were dehydrated in a graded ethanol series, and embedded in SPURR resin. To identify the antennal lobes (ALs), semi-thin coronal sections of the whole brains were cut with a Leica EM UC6 ultramicrotome, stained with toluidine blue and observed with a Leica DM2700 P light microscope. Sections of about 70 nm corresponding to portions of the ALs and thoracic ganglia were cut with a diamond knife on a Leica EM UC6 ultramicrotome. Transmission electron microscopy (TEM) images were collected with a FEI Tecnai G2 F20 (FEI Company, The Netherlands) and a Jeol JEM 1011 (Jeol, Japan) electron microscopes, working respectively at an acceleration voltage of 80 and 100 kV, and recorded with a 1 and 2 Mp charge-coupled device (CCD) camera (Gatan BM Ultrascan and GatanOrius SC100, respectively). T-bars density (expressed as number of T-bars/m²) in both ALs and thoracic ganglia presynaptic boutons was assessed on a total of ten animals (three WT, three untreated PINK1^B9 and four 0.1% Mpe-treated PINK1^B9 mutants). 459 and 683 T-bars were randomly sampled respectively in the ALs and the thoracic ganglia on a total 496 non-overlapping micrographs at a final magnification of 6000, corresponding to a total sampled area of more than 6000 µm². T-bars were unambiguously identified at presynaptic active zones by the presence of T-shaped electron-dense projections typically tethered by a large number of presynaptic vesicles.

The number of damaged mitochondria within ALs (expressed as percentage of the total number of mitochondria/sampled area) was evaluated in WT, untreated PINK1^B9 and 0.1% Mpe-treated PINK1^B9 mutants. More than 3000 mitochondria were randomly sampled on 191 non-overlapping micrographs at a final magnification of 4000, corresponding to a total sampled area of more than 5000 µm². Damaged mitochondria were recognized for the presence of swollen external membrane, clearly fragmented cristae and inhomogeneous electron transparent mitochondrial matrix. The mean differences were tested using a two tailed t-test and a p<0.01 level was considered statistically significant.

Group II WT, untreated PINK1^B9 and, as L⁺/A⁺, 0.1% Mpe- and 0.01% L-Dopa-treated PINK1^B9 mutants flies were collected and immediately stored at −80°C. Head lysate preparations of adult males were performed by homogenization in RIPA buffer (9.1 mmol/L dibasic sodium phosphate, 1.7 mmol/L monobasic sodium phosphate, 150 mmol/L sodium chloride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate [pH adjusted to 7.4]) containing fresh protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Two centrifugations were performed at 4°C at 10,000 g for 15 minutes, before protein quantification by DC Protein assay (Biorad, Hercules, CA, USA). 20 µg of proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis using the mini-PROTEIN 3-electrophoresis module assembly (Biorad, Hercules, CA, USA) and then transferred to immobilon-polyvinylidenedifluoride membranes (Amersham Biosciences). The membranes were incubated with primary antibodies overnight at 4°C. Immune complexes were detected with horseradish peroxidase–conjugated secondary antibodies and chemiluminescence reagents (ECL, Amersham Biosciences) and visualized by Image Quant LAS 4000. Densitometric analysis was performed by Image Studio Lite software for quantitative assessment.

Primary antibodies used in this study were against nc82 (1∶100 dilution, DSHB); Tyrosine Hydroxylase (1∶1000 dilution, MAB 318 Merk Millipore); actin (1∶100, sc1616 Santa Cruz Biotechnology); Horseradish-peroxidase–conjugated secondary antibodies were purchased from Life Technologies. Statistical significance of the results was evaluated by one-way ANOVA (p<0.05) followed by a HSD post-hoc test.

As shown in Fig. 1A, in agreement with our previous report [4], PINK1^B9 mutants displayed a significantly shorter lifespan with respect to WT flies. To assess the ability of Mpe to affect lifespan of PINK1^B9 mutants, they were supplied Mpe at different concentrations (0 (untreated), 0.1, 1 and 10% w/w in their standard diet) both as adults only (L⁻/A⁺) (Fig. 1B and Fig. S1A), and as larvae and adults (L⁺/A⁺) (Fig. 1C and Fig. S1B). The effects of L-Dopa (supplied as L⁺/A⁺ (at the concentration, 0.01%, at which is present in the Mpe 0.1%) on life span of PINK1^B9 are also reported in Fig. 1D. The comparison between untreated and Mpe-treated PINK1^B9, as shown by Kaplan-Meier survival curves, revealed a statistically significant effect of Mpe on lifespan of PINK1^B9 mutants only when L⁺/A⁺ flies were fed 0.1% Mpe (Fig. 1C, p<0.05 by Gehan-Breslow-Wilcoxon test). No effect was observed following the L-Dopa administration in L⁺/A⁺. As shown in Fig. S1A, no significant effects were detected in in L⁻/A⁺ flies, no matter the concentration tested, nor in L⁺/A⁺ flies fed 1% or 10% Mpe enriched standard diet (Fig. S1B).

To investigate the locomotor ability the negative geotaxis assay, as described previously [17], was used. An impairment of climbing behavior was observed in untreated PINK1^B9 at different age steps (I: 3–6; II: 10–15; III: 20–25 days old) with a worsening trend with aging, while WT flies fulfilled the evaluation criterion without differences among age groups. As shown in Fig. 2A, the mutants took longer times to accomplish the task than the WT (p<0.001).

The Mpe 0.1% treatment significantly ameliorated the climbing activity in mutants and also reduced the worsening trend with aging although the score obtained by treated mutants still remained higher than that measured in WT. Interestingly, the climbing time of L-Dopa-treated mutants from groups I and III did not significantly differ with respect to age-matched untreated PINK1^B9, the performance of only group II flies being significantly ameliorated.

As shown in Fig. 2B, L⁺/A⁺ 1% Mpe-treated mutants reached similar rescue of climbing activity as observed in 0.1% Mpe-treated ones only when tested at early ages (groups I and II). On the other hand, 10% Mpe administration failed to significantly ameliorate motor behavior in groups I and III with respect to untreated PINK1^B9 mutants, while a significant effect was detected in treated flies from group II. We also considered the percentages of flies that were able to complete the test and the results are depicted in histograms shown in Fig. 2C and D. In this respect, most of WTs of all age steps (97–98%) were able to complete the test, while only 76% of PINK1^B9 from group I, 46% from group II and 36% from group III accomplished it, showing a clear age-dependent worsening. Administration of 0.1% Mpe, as L⁺/A⁺, greatly rescued PINK1^B9 mutants (groups I–III) from motor impairment and restored to WT values the percentages (86–94%) of flies able to accomplish the task according to the evaluation criterion (10 sec). Furthermore, at variance with the above results, the effects of L-Dopa worsened over time. In particular, 0.01% L-Dopa administration determined a decrease of the number of flies able to complete the task showing a negative trend with aging. In fact, percentages of flies were 91%, 82% and 62%, in groups I, II and III, respectively.

As expected, the olfactory stimulations of flies’ antennae elicited responses with the typical EAG wave form, i.e. a rapid depolarization followed by a slower recovery phase, ending with the hyperpolarized wave before complete reversal to the baseline.

The results, summarized in Fig. 3A and 3B, show the olfactory response to 1-hexanol (0.01, 0.1 and 1%) elicited in WT, untreated PINK1^B9, 0.1% Mpe- and 0.01% L-Dopa-treated mutants from age group II. In details, the average EAG signal amplitudes evoked by stimuli were significantly lower in PINK1^B9 specimens in respect to WT thus substantially confirming data previously reported [4].

The stimulation with 1-hexanol at 1% did not elicit a significant increase in the EAG amplitude as compared with the stimulation at 0.1% in all strains of flies with the exception of mutants flies treated with L-Dopa 0.01%. This result indicates that at the highest odor concentrations (0.1 and 1%) a saturation of response was reached by all groups but by the L-Dopa treated mutants. Besides, we observed that the responses to stimuli in WTs elicited a greater hyperpolarized phase in the EAGs (Fig. 3B and S2).

Even if a positive trend in treated mutants exists in the signal amplitude in response to 1-hexanol, a statistical difference between untreated, Mpe- and L-Dopa-treated PINK1^B9 was not detected. The lowest odor concentration tested elicited a significantly higher response in WT as compared to the all strains of mutants (p<0.05). This difference shrank when the 0.1% concentration of odor was administered. A reduced response, although not statistically significant (p>0.05), was still detected in untreated mutants with respect to WTs (p<0.05), while treated flies showed on average an increased response with respect to untreated flies. The response measured in treated flies was therefore halfway between the highest of WTs and the lowest of untreated PINK1^B9. Samples of EAGs responses are shown in Fig. 3B and Fig. S2.

The olfactory behavior assay was restricted to flies of group II, by testing the responses to 1-hexanol (0.1% v/v) of WT, untreated PINK1^B9, 0.1% Mpe- and 0.01% L-Dopa-treated, as L⁺/A⁺, PINK1^B9 mutants. As expected, the analysis of the result, shown in Fig. 4, confirmed the olfactory behavioral impairment in PINK1^B9 flies [4]. In fact, only 29.6±4.4% of mutant flies were odor-trapped, while the percentage of baited WT (52.9±6.6%) was significantly higher (p<0.004). PINK1^B9 flies treated with 0.1% Mpe and 0.01% L-Dopa were able to reach the stimuli as WT controls (p>0.05). In fact, percentages of trapped flies were 45.2±5.8% and 44.2±3.6% for 0.1% Mpe- and 0.01% L-Dopa-treated mutants, respectively. Similar results were obtained concerning the numbers of trapped flies in the blank bait (H₂O) (p<0.05 between untreated PINK1^B9with respect to WT, 0.1% Mpe- and 0.01% L-Dopa-treated mutants).

Transmission electron microscopy (TEM) analysis was restricted to flies of group II of untreated WT, untreated PINK1^B9 and 0.1% Mpe-treated, as L⁺/A⁺, PINK1^B9 mutants and results are shown in Fig. 5. A significant decrease of T-bars density was observed in the presynaptic bouton active zones of both ALs and thoracic ganglia of PINK1^B9 mutants with respect to WT controls (panels A, B, E and F). More importantly, a significant increase of T-bars density was detected in the ALs and thoracic ganglia of PINK1^B9 treated with 0.1% Mpe, as L⁺/A⁺, with respect to untreated PINK1^B9 (panels A, B, E and F). Moreover the number of damaged, swollen and with clearly fragmented cristae, mitochondria was significantly lower in presynaptic boutons of ALs of PINK1^B9 mutants treated with 0.1% Mpe, compared with untreated mutants (panels C, D and G).

Fig. 6 shows the results of western blot analysis of whole brain expression of BRP and TH of flies of group II WT, untreated PINK1^B9 0.1% Mpe- and 0.01% L-Dopa-treated, as L⁺/A⁺, PINK1^B9 mutants. As shown in Fig. 6A, the expression of BRP and TH in untreated PINK1^B9 mutants was significantly lower (p<0.05) than in WT. Diet supply of 0.1% Mpe to PINK1^B9 mutants significantly recovered BRP and TH expression to WT controls levels (p<0.05) and these values did not differ statistically from those of WT. Notably, BRP and TH expression in PINK1^B9 mutants fed 0.01% L-Dopa resulted similar to BRP and TH expression in untreated PINK1^B9 mutants. ANOVA also revealed that both BRP and TH expression resulted statistically different as compared to their expression of both WT and PINK1^B9 mutants fed 0.1% Mpe.